Signalling pathway of U46619-induced vascular smooth muscle contraction in mouse coronary artery

Thromboxane A₂ (TXA₂ ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA₂ -induced contraction in the coronary artery remains to be clarified. A multi myograph system was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA₂ analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca²⁺ ]_i ) in mouse coronary artery smooth muscle cells. Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration-dependent manner, which was completely abolished by a specific TXA₂ receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but was not blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1 μmol/L nifedipine, then mostly inhibited by 100 μmol/L 2-APB and 10 μmol/L Y27632. We further found that the response to U46619 was inhibited, respectively, by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca²⁺ (SOC) channels generated the increase of [Ca²⁺ ]_i in mouse coronary artery smooth muscle cells, SOC channels did not contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca²⁺ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca²⁺ activated C1^- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca²⁺ ]_i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signalling pathway in TP downstream proteins, while SOC channels did not participate in the vasoconstriction.